In Vitro analysis of the H15-hexose symporter on the plasma membrane of sugarbeets (Beta vulgaris L)

Abstract

The mechanism of hexose transport into plasma membrane vesicles isolated from mature sugarbeet leaves (Beta vulgaris L.) was investigated. The initial rate of glucose uptake into the vesicles was stimulated approximately fivefold by imposing a transmembrane pH gradient (ApH), alkaline inside, and approximately four-fold by a negative membrane potential (Δψ), generated as a K15-diffusion potential, negative inside. The -fold stimulation was directly related to the relative ΔpH or Δψ gradient imposed, which were determined by the uptake of acetate or tetraphenylphosphonium, respectively. Δψ- and ΔpH-dependent glucose uptake showed saturation kinetics with a Km of 286 micromolar for glucose. Other hexose molecules (e.g. 2-deoxy-D-glucose, 3-O-methyl-D-glucose, and D-mannose) were also accumulated into plasma membrane vesicles in a ΔpH-dependent manner. Inhibition constants of a number of compounds for glucose uptake were determined. Effective inhibitors of glucose uptake included: 3-O-methyl-D-glucose, 5-thio-D-glucose, D-fructose, D-galactose, and D-mannose, but not 1-O-methyl-D-glucose, D- and L-xylose, L-glucose, D-ribose, and L-sorbose. Under all conditions of proton motive force magnitude and glucose and sucrose concentration tested, there was no effect of sucrose on glucose uptake. Thus, hexose transport on the sugarbeet leaf plasma membrane was by a H+-hexose symporter, and the carrier and possibly the energy source were not shared by the plasma membrane H+-sucrose symporter.